1. Field of the Invention
The present invention relates to test devices useful for performing homogeneous specific binding assays. It has been discovered that the accuracy of such devices in determining the presence of a ligand in, or the ligand binding capacity of, a liquid test sample can be significantly enhanced. Thus, assays can be performed with far greater reproducibility and accuracy than heretofore possible.
Accordingly, the present invention relates to the incorporation of homogeneous specific binding assay reagent systems into a dry, dip-and-read test device format. Because the discovery described herein enhances the accuracy of such a format, it is no longer necessary for such assays to be conducted using solutions of reagents, either premixed and stored, or mixed at the time of performing the assay.
Test devices in the form of test strips and similar solid state analytical elements have become commonplace in the analysis of various types of liquid samples such as biological fluids, industrial fluids, and others. Their popularity stems from among other reasons, their convenience and speed. As a result, test strips have been designed for detecting various clinically significant substances in both serum and urine. They have proven to be extremely advantageous in assisting the diagnosis and treatment of disease states in man and animals.
Test strip devices generally comprise a carrier member, such as an absorbent or bibulous matrix, incorporated with reagents which interact with one or more constituents of a liquid test sample to provide a detectable response, such as color change. The sample under assay is contacted with the reagent-incorporated carrier member, such as by momentarily immersing the device in the sample or by applying to it an aliquot of the sample, and the response is observed after a given time has elapsed. One great advantage of such test devices is the convenience of their routine use. They eliminate such inconvenient, time consuming steps as reagent additions to the sample and provide a rapid, easily observable signal.
Test strips of various types are known for use in a wide variety of fields. They include the familiar pH test paper devices, and a migriad of in vitro diagnostic devices for the detection of various urine and blood components such as glucose, protein, occult blood and so forth (e.g., as described in U.S. Pat. Nos. 3,164,534; 3,485,587; and 3,012,976). Reagent compositions found in such conventional test strips interact with the constituent or constituents to be determined by direct chemical or enzymatic reaction and, for this and other reasons, have limited sensitivity, being applied to the detection of substances that are present in liquid samples at concentrations in the millimolar range or above.
On the other hand, the development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between the ligand, i.e., the bindable analyte under determination, and a binding partner such as an antibody. Where one of the ligand or its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In conventional specific binding assay techniques, a sample of the liquid to be assayed is combined with reagent systems of various compositions. Such compositions include a labeled conjugate comprising a binding component incorporated with a label. The binding component in the labeled conjugate participates with other constituents, if any, of the reagent system and the ligand under assay to form a binding reaction system producing two species or forms of the labeled conjugate: a bound-species and a free-species. In the bound-species the binding component in the labeled conjugate, e.g., a hapten or antigen, is bound by a corresponding binding partner (e.g., an antibody) whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand to be detected in the test sample.
Where the labeled conjugate in the bound-species is essentially indistinguishable in the presence of the labeled conjugate in the free-species by the means used to monitor the label, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to in the art as "heterogeneous". Where the bound-species and free-species forms of the labeled conjugate can be distinguished in the presence of each other, the separation step can be avoided, and the assay is said to be "homogeneous".
The radioimmunoassay was the first discovered type of highly sensitive specific binding assay to employ a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials and the necessity of a separation step, homogeneous assay systems have been devised using materials other than radioisotopes as the label component, incuding enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme activators and inhibitors, cycling reactants, organic and inorganic catalysts, prosthetic groups, chemiluminescent reactants, and fluorescent molecules. Such homogeneous specific binding assay systems provide a detectable response, e.g., an electromagnetic radiation signal, such as a color change, chemiluminescence, or fluorescence emission, which is related to the presence or amount of the ligand under assay in the liquid sample.
Commercially available test means for performing specific binding assays are usually in the form of test kits comprising a packaged combination of containers holding solutions or rehydratable compositions of the reagents necessary for carrying out the assay. To perform the actual assay method, aliquots of such solutions must be manually or instrumentally dispensed into a reaction vessel with the sample. If manually dispensed, the assay consequently requires the time and skill of a technician, and if instrumentally dispensed, the assay consequently requires the expense and maintenance of dispensing apparatus.
2. Brief Description of the Prior Art
The problem of instability encountered with apoglucose oxidase stands in direct contrast to the marked stability experienced with the corresponding holoenzyme glucose oxidase. Swoboda, Biochem. Biophys. Acta, 175, pages 365-379 (1969). When glucose oxidase is heated to 50.degree. C. for 10 minutes it retains 98% of its enzyme activity; similar treatment of the apoenzyme results in only 20% enzyme activity upon reconstitution with FAD (flavin adenine dinucleotide). Similarly, proteolytic digestion experiments with pronase-P at 0.degree. C. for 1.5 hours yielded 98% retained activity for the holoenzyme, whereas the apoenzyme retained only 21% activity. The prior art appears bereft of attempts to stabilize the apoenzyme; only the problem of instability has been recognized.
It is known that glutathione synthetase in very dilute solutions is relatively unstable, and it was suggested that bovine serum albumin could be used to stabilize enzyme in such dilute solutions. S. P. Colowick, et al., eds., "Methods in Enyzmology", Volume II, pages 342-346, Academic Press, Inc., New York (1955). See also Ibid., Volume IV, page 367 (1957).
Solid phase test devices have been applied to heterogeneous specific binding assays in attempts to overcome the inconveniences and disadvantages of the requisite separation step. A commonly used solid phase device of this type comprises a nonporous surface, such as the interior surface of a test tube or other vessel, to which antibody is affixed or coated by adsorption or covalent coupling. U.S. Pat. Nos. 3,826,619; 4,001,583; 4,017,597; and 4,105,410 relate to the use of antibody coated test tubes in radioimmunoassays. Solid phase test devices have also been used in heterogeneous enzyme immunoassays (U.S. Pat. Nos. 4,016,043 and 4,147,752) and in heterogeneous fluorescent immunoassays (U.S. Pat. Nos. 4,025,310 and 4,056,724; and British Pat. Spec. No. 1,552,374).
The use of such heterogeneous specific binding assay test devices is exemplified by the method of U.S. Pat. No. 4,135,884 relating to a so-called "gamma stick." The test device is incorporated with the antibody reagent and is brought into contact with the liquid sample and with the remaining reagents of the reaction system, principally the labeled conjugate. After an incubation period, the solid phase device is physically removed from the reaction solution and the label measured either in the solution or on the test device.
Similar devices where the antibody reagent is entrapped in a matrix such as a gel or paper web are described in U.S. Pat. Nos. 3,925,017; 3,970,429; 4,138,474; 3,966,897, 3,981,981 and 3,888,629 and in German OLS No. 2,241,646. Likewise, devices for use in heterogeneous specific binding assays wherein the antibody reagent is fixed to a matrix held in a flowthrough column are known (U.S. Pat. Nos. 4,036,947; 4,039,652; 4,059,684; 4,153,675; and 4,166,102). As with all heterogeneous specific binding assay devices, the test device is usually incorporated with less than all of the necessary reagents for carrying out the assay and is merely a means for rendering more convenient the necessary separation step.
Finally, heterogeneous specific binding assay test devices have been described wherein most or all of the necessary reagents are incorporated with the same carrier element, and wherein reagent/sample contacts and separation of the free- and bound-phases are accomplished by capillary migrations along the carrier element (U.S. Pat. Nos. 3,641,235; 4,094,647 and 4,168,146). The devices described in such patents are generally considered difficult to manufacture and susceptible to irreproducibility due to the complex nature of the many chemical and physical interactions that take place along the carrier element during performance of an assay and the low concentrations of ligand or analyte which such devices are intended to determine.
The application of homogeneous specific binding assay reagent systems to solid state test devices would provide great advantages to the routine user of such assay systems. The determination of ligands appearing in very low concentrations in liquid samples would be simplified to the steps of contacting the device with the sample and measuring, either by visual observation or by instrumental means, the resulting signal. Reagents would be provided in a solid form, with no need to store, dispense or mix liquid reagents as is required when using the prior art test kits. Solid state devices would also be much more adaptable to automation than the prior art liquid systems.
The prior art lacks a detailed teaching of how to apply homogeneous specific binding assay reagent systems to solid state test devices. British Pat. Spec. No. 1,552,607, commonly assigned herewith, describes homogeneous specific binding assay systems employing various novel labels, including chemiluminescent labels, enzyme substrate labels and coenzyme lables. At page 23, lines 12 et seq of this patent there is the suggestion to incorporate the assay reagents with various carriers including liquid-holding vessels or insoluble, porous, and preferably absorbent, matrices such as bibulous papers; polymeric films, membranes, fleeces, or blocks; gels; and the like.
German OLS No. 2,537,275 describes a homogeneous specific binding assay reagent system and poses the possibility of using slides or strips incorporated with antibody in performing the assay. In this suggestion, the labeled conjugate would be first mixed with the sample and thereafter the antibody incorporated test device contacted with the reaction mixture. After a suitable incubation time, it is proposed that the test device would be rinsed with buffer, dried, and then the signal (fluorescence) measured. Thus, this German OLS poses a test device and assay method much like those already known for heterogeneous specific binding assay techniques wherein the test device is immersed in the liquid reaction mixture, incubated, thereafter removed, washed, and finally read. Additionally, the proposed test device does not incorporate all of the binding assay reagents with the carrier element. Specifically, only the antibody is proposed to be incorporated with the carrier element with the labeled conjugate being separately added to the sample under assay prior to contact with the proposed test device.